ENDOTOXIN REMOVAL

Endotoxin is a complex aggregate of acidic lipopolysaccharides (LPS) and consists of an innermost core of hydrophobic fatty acid groups and a central and outermost region composed of hydrophilic polysaccharides.

In aqueous solutions, endotoxin can exist in various states of aggregation up to 1 MDa. Divalent cations, such as Ca2+ and Mg2+, are found to stabilize the aggregated structure of LPS, whereas detergents help to break down the structure into smaller sub-units. When producing recombinant proteins in E. coli and other gram-negative bacteria, it is often necessary to remove LPS from the final product. This is especially important when carrying out immunological readouts and when developing manufacturing processes. Endotoxin can cause false readings in cell-based assays. There are limits to the amounts of endotoxin allowed in human products.

Endotoxin is continuously shed from the outer membrane of viable gram-negative bacteria and is released when the bacterial cell dies. Although bacteria are often removed by using a 0.2 μm sterilizing grade filter, LPS itself is difficult to remove or inactivate because it is extremely heat and pH stable. The pyrogenic threshold of an endotoxin reaction is on the order of 1 EU (endotoxin unit ~0.1 ng) per kg of body weight. This amount of endotoxin can come from 105 bacterial cells.

The removal of endotoxin is one of the most difficult downstream processes during protein purification. Many commercially available products are unable to remove endotoxin satisfactorily, or require time-consuming incubation steps. In many cases, complete endotoxin removal is only achieved with massive substrate loss. Because endotoxin is negatively charged at pH above 2, a positively- charged membrane surface can remove endotoxin. 

The Acrodisc® unit with positively-charged, hydrophilic Mustang® E membrane is ideal for the removal of endotoxin from solution due to its highly crosslinked quaternized amine charged surface. This gives very high dynamic capacities under selected conditions for the removal of endotoxin from process feedstreams, buffers, and water.

When using E. coli or other gram negative bacteria for recombinant proteins, endotoxin is likely to contaminate the plasmid preparation and the expressed protein. Before conducting additional assays, such as immunologic readouts or developing a manufacturing process, this complex lipopolysaccharide (LPS) should be removed. While intact bacteria can be captured using a 0.2 µm filter, the LPS can be more challenging. An effective method of removal of the negatively charged LPS is to use a substrate with a positive charge. We offer Acrodisc® Units with Mustang® E Membrane for effective removal of endotoxin.